RU2264826C1 - Method for obtaining antithymocytic globulin for intravenous injection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the present innovation deals with immunobiological medicinal preparations applied for intravenous injection at correcting immunodeficient states of different genesis. One should immunize animals with human thymus gland's cells, isolation of immune plasma, its depletion with plasmatic proteins AB (IV) human blood group followed by plasmatic fractioning with PEG 6000 and 20000, depleting semi-product with cellular components of the mixture of all four human blood groups and purification of the target product with PEG 6000. As a stabilizing agent on should apply glycine.

EFFECT: more simplified method.

4 cl, 3 ex

Description

The invention relates to medicine, namely to the production of immunobiological drugs used for intravenous administration in the correction of immunodeficiency states of various origins.

A known method of producing immunoglobulin from biological fluids by adsorption on activated carbon [1].

The disadvantage of this method is the low capacity of the coal in relation to immunoglobulins and the low selectivity of the specified sorbent, which ensures the release of not more than 1% of immunoglobulins from the total amount of adsorbed protein.

There is also a method of extracting immunoglobulins from biological fluids by passing them through sorbents, which are used as a macroporous copolymer of styrene and divinylbenzene containing groups of asymmetric dimethylene-bis-ammonium bases [2].

The disadvantage of this method is its complexity and low degree of purification, which does not allow the use of the drug for intravenous administration due to its inconsistency with modern requirements for such drugs.

A known method of producing immunoglobulins by fractionation of blood proteins using ethanol [3].

The disadvantage of this method is the need to use high concentrations of ethanol, creating a risk of protein denaturation, which makes it necessary to carry out the process at low temperatures.

Another disadvantage of this method is the need to use ion exchange chromatography followed by ultrafiltration for additional purification of immunoglobulins from impurities of ballast proteins and ethanol, which significantly complicates the process, affects the quality of the drug and increases its cost.

Closest to the claimed invention is a method for the preparation of antilymphocytic globulin, based on the preparation of blood plasma from animals (for example, rabbits, goats, horses) immunized with thymus cells of a human embryo by fractionating it with rivanol to release from ballast proteins, followed by adsorption of plasma heteroagglutinins on erythrocytes AB (IV) of a group of human donated blood and post-treatment of an intermediate using polyethylene glycol (PEG) 6000 [4].

The main disadvantage of this method is the complexity of the rivanol fractionation process and the need for further purification of the intermediate from this reagent.

In addition, the production of rivanol in Russia is currently discontinued, and the cost of a foreign reagent is very high, which makes it inaccessible for use in the large-scale production of an antithymocytic globulin preparation.

The objective of the invention is to improve the method of producing highly purified areactogenic antithymocytic globulin (ATG) for intravenous administration at the scale necessary for health care needs.

The technical result of the use of the invention is the development of a high-tech large-scale method for producing ATG, which fully complies with the requirements for similar drugs for intravenous administration and is used to prevent and stop crises of rejection of organ transplants, in particular, kidneys, liver, as well as for the treatment of aplastic anemia, struggle with purulent-inflammatory conditions, etc.

The essence of the proposed method for producing ATG for intravenous administration is that animals, for example goats, rabbits, horses, are immunized with thymus cells of a human embryo, the plasma of immunized animals is isolated, their depletion is carried out by human plasma of group AB (IV), adding the latter in a ratio of 20 -25 / 1-1.5 (v / v), respectively. Then plasma proteins are fractionated using PEG 6000, and antibodies to non-lymphoid antigens are removed from the fractionated mixture by sequential treatment with a mixture of washed donor red blood cells and platelet concentrate from four human blood groups. Next, the target product is purified by precipitation of PEG 6000, adding it to a final concentration of 10-12% (w / v) and subsequent centrifugation. The resulting protein precipitate, consisting of immunoglobulins, is dissolved with physiological saline, glycine is added as a stabilizer, clarifying and sterilizing filtration, bottling and lyophilization of the preparation are carried out. At the same time, fractionation of depleted plasma proteins is carried out in three stages: at the first stage, the target product PEG 6000 is precipitated, pH 7.0-7.2, at its final concentration of 10-12% weight / volume, at the second stage, the precipitated product is suspended in distilled water and high molecular weight proteins are removed by precipitation with their PEG of 20,000 at a final concentration of 2-2.5% weight / volume, pH 5.4, followed by centrifugation and, in the third step, the target product is reprecipitated from the fractionated PEG 6000 mixture at a final concentration of 10-12 % weight / volume, pH 7.0-7.2.

The method is as follows. Animals, for example, goats, rabbits, horses, are repeatedly immunized with a suspension of human thymus gland cells taken from the fetus of a person who died as a result of asphyxiation or birth injury (the first immunization uses Freund's complete adjuvant). 2 weeks after the last immunization, blood is drawn from animal producers followed by the separation of the immune plasma. To the obtained plasma add the blood plasma of human blood of the AB (IV) group in a ratio of 20-25: 1-1.5, respectively. Then the fractionation of the proteins of the depleted plasma is carried out in three stages: at the first stage, the target product PEG 6000, pH 7.0-7.2, is precipitated, adding it to a final concentration of 10-12% (w / v) and low-speed centrifugation at 3000 rpm min for 10 minutes in a HERMLE ZK 630 centrifuge. Then, to a precipitate suspended in distilled water, add PEG 20,000, pH 5.4, to a final concentration of 2-2.5% (w / v), centrifuge and separate the precipitate of high molecular weight proteins. At the third stage, to the supernatant, the pH of which is adjusted to 7.0-7.2, PEG 6000 is added to a final concentration of 10-12% (w / v), centrifugation is carried out, the supernatant is removed, and the immunoglobulin precipitate is dissolved in physiological saline. Antibodies to non-lymphoid antigens are sequentially depleted with a mixture of red blood cells and platelets of four human blood groups and centrifuged. The target product is precipitated using PEG 6000, adding it to a final concentration of 10-12% (weight / volume). After centrifugation at 3000-3500 rpm for 10-15 minutes, a protein precipitate, which is (at least 90%) of class G and M immunoglobulins, is dissolved with physiological saline, glycine (15-25 g / l of dissolved protein) is added as a stabilizer ), then conduct clarifying and sterilizing filtration, bottling and subsequent lyophilization. The target product is dissolved in physiological saline before use. The activity of the drug in the lymphocytotoxic test (LCTT) is at least 1024-2048.

Example 1. Healthy goats of both sexes aged 1-3 years weighing 18-30 kg are immunized 6 times with a suspension of cells obtained from the thymus glands of the fetus or the corpses of newborn babies who died as a result of asphyxiation or birth injury in the first days after delivery. At the first immunization of animals, the antigen is administered simultaneously with Freund's complete adjuvant. A mixture consisting of 2 ml of suspension of human thymus gland cells at a concentration of 3 × 10 ⁸ and 2 ml of adjuvant is administered intramuscularly to animals, 1 ml at four points. After two weeks, 5 subsequent immunizations are carried out with an interval of 3-5 days. Each animal is injected with 2 ml of antigen. Two weeks after the last immunization, a blood preparation is made with a blood preservative, which eliminates the hemolysis of red blood cells. Immune plasma is separated from blood cells by centrifugation. Then this plasma is treated with plasma of human blood donation of the AB (IV) group in a ratio of 20: 1. The mixture is thoroughly mixed for at least 30 minutes. Then carry out the selection and purification of the target product in three stages. At the first stage, the target product PEG 6000, pH 7.0-7.2, is precipitated, adding it to a final concentration of 10% (weight / volume) and performing low-speed centrifugation at 3000 rpm for 10 minutes in a HERMLE ZK 630 centrifuge. At the second stage, to the precipitated product suspended in distilled water, PEG 20,000 is added to a final concentration of 2.5% (w / v), the pH is adjusted to 5.4, centrifuged under the same conditions and to remove high molecular weight proteins. At the third stage, to the supernatant, the pH of which is adjusted to 7.0-7.2, PEG 6000 is added to a final concentration of 10% (weight / volume), centrifuged at 3000 rpm for 10 minutes, the supernatant is removed, dissolved precipitate of immunoglobulins in saline.

To release the fractionated mixture from antibodies to non-lymphoid antigens, a mixture of washed donor red blood cells and platelet concentrates of four human blood groups is added sequentially. A suspension of washed red blood cells is added in a ratio of 0.75: 1 (v / v), mixed thoroughly several times. After a 10-minute exposure, the mixture is centrifuged for 10 min at 2500 rpm at 10 ° C, and then human platelets are added at the rate of 1.75 ml / l of the original plasma, left for 30 minutes at room temperature and periodically stirred, after which centrifuged for 20 min at 3900 rpm at a temperature of 10 ° C. The supernatant is sterile collected and the desired product is isolated using PEG 6000, which is added to a final concentration of 11% (w / v) with continuous stirring for 30 minutes. Then the mixture is centrifuged for 10 min at 3000 rpm, the supernatant is removed, and physiological sodium chloride solution is added to the protein precipitate until the precipitate is completely dissolved. The total protein content in the intermediate, determined, for example, by the biuret method, is 10-15 mg / ml. Then glycine is added as a stabilizer at the rate of 20 g / l of dissolved protein, the pH is adjusted to 7.3-7.5, and then the drug is clarified and sterilized through membrane filters of the Millipor type with the corresponding pore sizes, filling the drug into ampoules and its lyophilization.

In one ampoule of the target product with a volume of 3-5 ml contains 50 ± 10 mg of protein, which, when controlled by electrophoresis on cellulose acetate films, consists of more than 90% immunoglobulins. This degree of purification complies with national and international requirements for immunoglobulins for intravenous administration. The drug does not contain preservatives and antibiotics, its activity in LCTT is at least 1: 1024-2048.

Example 2. Obtained by the proposed method, the drug ATG (commercial name Antilimfolin) was used to treat a patient of Peninsula 34 years (medical history No. 748). Diagnosis: acute hemorrhagic pancreatic necrosis, extensive retroperitoneal phlegmon, peritonitis. Concomitant diseases: left-sided hydrothorax, hydropericardium.

Despite all the generally accepted methods of treatment, modern complex therapy, the general condition of the patient remained very difficult. The immunogram indicated a sharp decrease in the immune status of the patient. On the 2nd day after the administration of Antilimpholine, the phagocytic activity of neutrophils (FAN) significantly increased. On the 7th day after the use of the drug, the number of immunocompetent cells significantly increased (2-3 times), a further increase in FAN was noted; an increase in the number of immunoglobulins G and M was observed. The use of Antilimfolin had an immunostimulating effect and contributed to a significant improvement in the condition of the patient, who was soon transferred from the intensive care unit to the general surgery ward with subsequent discharge home ..

Example 3. Patient K., 23 years old (medical history No. 4819), was admitted to the clinic with complaints of general weakness, shortness of breath during physical exertion, nose and gingival bleeding. In production, had contact with benzene compounds. Blood test at admission: Hb - 47 g / l, red blood cells 1.5 · 10 ¹² / l, white blood cells 2.6 · 10 ⁹ / l, segmented nuclear - 12% (0.312 · 10 / l), lymphocytes 87% (2, 26 · 10 ⁹ / l), platelets 6 · 10 ⁹ / l. In bone marrow puncture: myelocaryocytes 9.0 · 10 ⁹ / l, immature granulocytes 7.5%, mature 17.5%, lymphocytes 70.5%, erythronormoblasts 4.5%. In trepanobioptate - aplasia of hematopoiesis. Only small accumulations of red blood cells, lymphocytes and single siderophages were detected. In the immunogram: CD ₃ ⁺ CD ₈ ⁺ - 47% (0.9 · 10 ⁹ / l), CD ₃ ⁺ CD ₄ ⁺ - 40.7% (0.8 · 10 ⁹ / l), CD ₂₀ ⁺ 2, 5% (0.05 · 10 ⁹ / l), CD ₃ ⁺ CD ₁₆ ⁺ - 18.6% (0.4 · 10 ⁹ / l). MDC in the bone marrow: erythroid row - 20%, granulocytic row - 40%, lymphoid row - 40%. MDC in the peripheral blood: erythrocytes - 36%, granulocytes - 15%, lymphocytes - 30%. Within 5 days, the patient received 15 mg / kg of body weight of antilimpholine. The total dose was 4.62 g of protein. On the third day of the drug administration, the temperature rose to 38 ° C, skin petechiae appeared, hemorrhagic manifestations intensified. Additionally, antihistamine therapy was performed. Subsequently, blood transfusion, hemostatic, antibacterial therapy was added in connection with the emerging infectious complications (right-sided pneumonia, acute pyelonephritis). 1.5 months after treatment, clinical remission was achieved and the patient was discharged home. Blood test at discharge: Hb - 86 g / l, erythrocytes - 2.7 · 10 ¹² / l, white blood cells - 3.0 · 10 ⁹ / l, segmented - 31%, lymphocytes - 65%, platelets - 7.0 · 10 ⁹ / l. There are no hemorrhagic manifestations. The dynamics of peripheral blood and bone marrow was positive. During the observation period, clinical and hematological remission persists. The patient is studying and working.

LITERATURE

1. Lopukhin Yu.M., Molodenkov M.N. Hemosorption. - M. - Medicine. - 1978. - S. 31-34; 139-141.

2. USSR copyright certificate No. 1121619 of March 29, 83.

3. USSR author's certificate No. 1403414 of 02.15.88.

4. RF patent No. 2178309 dated 01/12/2000.

Claims (5)

1. A method of producing anti-thymocyte globulin for intravenous administration, comprising immunizing animals with human thymus cells, depleting the resulting plasma by treating it with human plasma of the AB (IV) group, fractionating plasma proteins, removing from the fractionated mixture of antibodies to non-lymphoid antigens by sequential treatment with a mixture of washed donor red blood cells and platelet concentrate of four human blood groups, purification of the target product by precipitation of PEG 6000, dissolution of the obtained immunoglobulin in physiological saline, the addition of glycine and clarifying and sterilizing filtration, characterized in that the fractionation of depleted plasma proteins is carried out in three stages: at the first stage, the target product PEG 6000, pH 7.0-7.2, is precipitated, the precipitated product is suspended in distilled water and impurities are removed by low-speed centrifugation, in the second step, high molecular weight proteins are removed from the precipitated product by precipitation with their PEG 20,000, pH 5.4, and in the third step, the target product is reprecipitated from the fractionated mixture and PEG 6000, pH 7.0-7.2. 2. The method according to claim 1, characterized in that the deposition of the target product from depleted plasma at the first stage of fractionation is carried out by PEG 6000 at a final concentration of 10-12% weight / volume. 3. The method according to claim 1, characterized in that the high molecular weight proteins in the second stage precipitate PEG 20,000, pH 5.4 at a final concentration of 2-2.5% weight / volume. 4. The method according to claim 1, characterized in that the re-precipitation of the target product from the fractionated mixture in the third stage is carried out at a final concentration of PEG 6000 of 10-12% weight / volume. 5. The method according to claim 1, characterized in that after sterilizing and clarifying filtration, the resulting antithymocytic globulin is lyophilized.